Oral administration of TRPV4 inhibitor improves atrial calcium handling abnormalities in sterile pericarditis rats / 生理学报

Atrial Ca2+ handling abnormalities, mainly involving the dysfunction of ryanodine receptor (RyR) and sarcoplasmic reticulum Ca2+-ATPase (SERCA), play a role in the pathogenesis of atrial fibrillation (AF). Previously, we found that the expression and function of transient receptor potential vanilloid subtype 4 (TRPV4) are upregulated in a sterile pericarditis (SP) rat model of AF, and oral administration of TRPV4 inhibitor GSK2193874 alleviates AF in this animal model. The aim of this study was to investigate whether oral administration of GSK2193874 could alleviate atrial Ca2+ handling abnormalities in SP rats. A SP rat model of AF was established by daubing sterile talcum powder on both atria of Sprague-Dawley (SD) rats after a pericardiotomy, to simulate the pathogenesis of postoperative atrial fibrillation (POAF). On the 3rd postoperative day, Ca2+ signals of atria were collected in isolated perfused hearts by optical mapping. Ca2+ transient duration (CaD), alternan, and the recovery properties of Ca2+ transient (CaT) were quantified and analyzed. GSK2193874 treatment reversed the abnormal prolongation of time to peak (determined mainly by RyR activity) and CaD (determined mainly by SERCA activity), as well as the regional heterogeneity of CaD in SP rats. Furthermore, GSK2193874 treatment relieved alternan in SP rats, and reduced its incidence of discordant alternan (DIS-ALT). More importantly, GSK2193874 treatment prevented the reduction of the S2/S1 CaT ratio (determined mainly by RyR refractoriness) in SP rats, and decreased its regional heterogeneity. Taken together, oral administration of TRPV4 inhibitor alleviates Ca2+ handling abnormalities in SP rats primarily by blocking the TRPV4-Ca2+-RyR pathway, and thus exerts therapeutic effect on POAF.

Combined transgenic inhibition of CaMKII and Ik1 on cardiac remodeling / 生理学报

This study was aimed to establish an experimental mouse model of combined transgenic inhibition of both multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and inward rectifier potassium current (Ik1), and to observe whether the specific inhibition of both CaMKII and Ik1 can bring about any effects on cardiac remodeling. Mice were divided into 4 groups: wild type (WT), CaMKII inhibited (AC3-I), Ik1 inhibited (Kir2.1-AAA) and combined inhibition of both CaMKII and Ik1 (AC3-I+Kir2.1-AAA). Mice in each group received electrocardiogram (ECG) and echocardiography examination. ECG in the condition of isoproterenol (ISO) injection was also checked. The whole cell patch clamp technique was used to measure Ik1 and the transient outward potassium current (Ito) from enzymatically isolated myocytes of left ventricle. In the condition of basal status, no significant changes of heart rate, PR interval and QRS interval were observed. No mouse showed ventricular arrhythmias in all of the 4 groups. After ISO injection, each group presented no significant ventricular arrhythmias either. The indexes measured by M-mode (motion-mode) and two-dimensional echocardiography had no significant differences among the four groups. Ik1 in AC3-I group was significantly higher than those in other three groups (P < 0.01) because of the results brought about by CaMKII inhibition. Among the latter three groups, both Kir2.1-AAA group and AC3-I+Kir2.1-AAA group had a significant reduced Ik1 compared with that of WT group, which was due to the Ik1 inhibition (P < 0.01). Ito in AC3-I group was higher than that of the other three groups (P < 0.01), but there were no significant differences in Ito among WT, Kir2.1-AAA and AC3-I+Kir2.1-AAA groups. Thus, combined transgenic myocardial CaMKII and Ik1 inhibition eliminated the up-regulation of Ik1 in CaMKII inhibited mice, and had no effects on cardiac remodeling including heart structure and function as well as arrhythmias at the basic and ISO conditions. The results of this study may provide a basis for the further investigation of combined inhibition of CaMKII and Ik1 in pathogenic cardiac remodeling.

Effect of Kv1.3 and KCa3.1 potassium ion channels on the proliferation and migration of monocytes/macrophages / 生理学报

This study was aimed to investigate the effects of blockade of Ca(2+) activated channel KCa3.1 and voltage-gated potassium channel Kv1.3 of the monocytes/macrophages on inflammatory monocyte chemotaxis. Chemotaxis assay was used to test the inflammatory Ly-6C(hi) monocyte chemotaxis caused by the monocytes/macrophages. The proliferation of monocytes/macrophages was detected by cell counting kit-8 (CCK8). Enzyme-linked immunosorbent assay (ELISA) was applied to detect the C-C motif ligand 7 (CCL7) in cultured media. The results showed that the recruitment of Ly-6C(hi) monocyte induced by monocytes/macrophages was suppressed by the potent Kv1.3 blocker Stichodactyla helianthus neurotoxin (ShK) or the specific KCa3.1 inhibitor TRAM-34. Meanwhile, the proliferation of monocytes/macrophages was significantly inhibited by ShK. The response of Ly-6C(hi) monocyte pretreated with ShK or TRAM-34 to CCL2 was declined. These results suggest that KCa3.1 and Kv1.3 may play an important role in monocytes/macrophages' proliferation and migration.

Role of voltage-gated potassium channels in pathogenesis of chronic pulmonary heart disease / 华中科技大学学报（医学）（英德文版）

The influence of hypoxia on the activity of voltage-gated potassium channel in pulmonary artery smooth muscle cells (PASMCs) of rats and its roles in the pathogenesis of chronic pulmonary heart disease were investigated. Eighty male Sprague-Dawley rats were randomly allocated into control group (n=10), acute hypoxic group (n=10), and chronic hypoxic groups (n=60). The chronic hypoxic groups were randomly divided into 6 subgroups (n=10 each) according to the chronic hypoxic periods. The rats in the control group were kept in room air and those in acute hypoxic group in hypoxia environmental chamber for 8 h. The rats in chronic hypoxic subgroups were kept in hypoxia environmental chamber for 8 h per day for 5, 10, 15, 20, 25, and 30 days, respectively. The mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy index (RVHI), and the current of voltage-gated potassium channel (I K) in PASMCs were measured. Results showed that both acute and chronic hypoxia could decrease the I K in PASMCs of rats and the I-V relationship downward shifted to the right. And the peak I K density at +60mV decreased with prolongation of hypoxia exposure. No significant difference was noted in the density of I K (at +60 mV) and I-V relationship between control group and chronic hypoxic subgroup exposed to hypoxia for 5 days (P>0.05), but there was a significant difference between control group and chronic hypoxic subgroup exposed to hypoxia for 10 days (P<0.05). Significant differences were noted in the I K density (at +60 mV) and I-V relationships between control group and chronic hypoxic subgroups exposed to hypoxia for 20 days and 30 days (P<0.01). Compared with control rats, the mPAP and RVHI were significantly increased after chronic exposure to hypoxia for 10 days (P<0.05), which were further increased with prolongation of hypoxia exposure, and there were significant differences between control group and chronic hypoxic subgroups exposed to hypoxia for 20 days and 30 days (P<0.01). Both the mPAP and the RVHI were negatively correlated with the density of I K (r=-0.89769 and -0.94476, respectively, both P<0.01). It is concluded that exposure to hypoxia may cause decreased activity of voltage-gated potassium channel, leading to hypoxia pulmonary vasoconstriction (HPV). Sustained HPV may result in chronic pulmonary hypertension, even chronic pulmonary heart disease, contributing to the pathogenesis of chronic pulmonary heart disease.

Role of voltage-gated potassium channels in pathogenesis of chronic pulmonary heart disease / 华中科技大学学报（医学）（英德文版）

The influence of hypoxia on the activity of voltage-gated potassium channel in pulmonary artery smooth muscle cells (PASMCs) of rats and its roles in the pathogenesis of chronic pulmonary heart disease were investigated. Eighty male Sprague-Dawley rats were randomly allocated into control group (n=10), acute hypoxic group (n=10), and chronic hypoxic groups (n=60). The chronic hypoxic groups were randomly divided into 6 subgroups (n=10 each) according to the chronic hypoxic periods. The rats in the control group were kept in room air and those in acute hypoxic group in hypoxia environmental chamber for 8 h. The rats in chronic hypoxic subgroups were kept in hypoxia environmental chamber for 8 h per day for 5, 10, 15, 20, 25, and 30 days, respectively. The mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy index (RVHI), and the current of voltage-gated potassium channel (I K) in PASMCs were measured. Results showed that both acute and chronic hypoxia could decrease the I K in PASMCs of rats and the I-V relationship downward shifted to the right. And the peak I K density at +60mV decreased with prolongation of hypoxia exposure. No significant difference was noted in the density of I K (at +60 mV) and I-V relationship between control group and chronic hypoxic subgroup exposed to hypoxia for 5 days (P>0.05), but there was a significant difference between control group and chronic hypoxic subgroup exposed to hypoxia for 10 days (P<0.05). Significant differences were noted in the I K density (at +60 mV) and I-V relationships between control group and chronic hypoxic subgroups exposed to hypoxia for 20 days and 30 days (P<0.01). Compared with control rats, the mPAP and RVHI were significantly increased after chronic exposure to hypoxia for 10 days (P<0.05), which were further increased with prolongation of hypoxia exposure, and there were significant differences between control group and chronic hypoxic subgroups exposed to hypoxia for 20 days and 30 days (P<0.01). Both the mPAP and the RVHI were negatively correlated with the density of I K (r=-0.89769 and -0.94476, respectively, both P<0.01). It is concluded that exposure to hypoxia may cause decreased activity of voltage-gated potassium channel, leading to hypoxia pulmonary vasoconstriction (HPV). Sustained HPV may result in chronic pulmonary hypertension, even chronic pulmonary heart disease, contributing to the pathogenesis of chronic pulmonary heart disease.

Effect of matrine on human ether à go-go related gene (HERG) channels expressed in Chinese hamster ovary cells / 中国结合医学杂志

<p><b>OBJECTIVE</b>To observe the effect of matrine on human ether à go-go related gene (HERG) potassium channels expressed in Chinese hamster ovary (CHO) cells and investigate whether HERG channel is a new target of the pharmacological effect of matrine on arrhythmia and tumor</p><p><b>METHODS</b>HERG channel potassium current in CHO cell was recorded using whole-cell patch-clamp technique, and the influence of matrine on the current was explored.</p><p><b>RESULTS</b>Matrine inhibited HERG potassium current in a dose-dependent manner, and the 50% inhibitory concentration (IC IC(50)) was 411±23 μmol/L. Matrine had no significant effect on the activation kinetics, and mainly blocked HERG channels in their closed state.</p><p><b>CONCLUSIONS</b>The blocking effect of matrine on HERG channels might be one of the mechanisms against arrythmias and tumors. Unlike most other blockers exerting blocking effect at the intracellular sites by entering the cell with the opening of HERG channel, matrine blocked HERG channels at the extracellular sites.</p>

Blockade of the human ether-a-go-go-related gene potassium channel by ketanserin / 生理学报

In the present study, we investigated the inhibitory action of ketanserin on wild-type (WT) and Y652 mutant human ether-a-go-go-related gene (HERG) potassium channels expressed in Xenopus oocytes and the effects of changing the channel molecular determinants characteristics on the blockade with and without ketanserin intervention using standard two-microelectrode voltage-clamp techniques. Point mutations were introduced into HERG gene (Y652A and Y652R) and subcloned into the pSP64 plasmid expression vector. Complementary RNAs for injection into oocytes were prepared with SP6 Cap-Scribe after linearization of the expression construct with EcoR I. Clampfit 9.2 software was employed for data collection and analysis. Origin 6.0 software was used to fit the data, calculate time constants and plot histograms. The results showed that ketanserin blocked WT HERG currents in voltage- and concentration-dependent manner and showed minimal tonic blockade of HERG current evaluated by the envelope of tails test. The IC50 value was (0.38+/-0.04) micromol/L for WT HERG potassium channel. The peaks of the I-V relationship for HERG channel suggested a negative shift in the voltage-dependence of activation after using ketanserin, whose midpoint of activation values (V1/2) were (-16.59+/-1.01) mV (control) vs (-20.59+/-0.87) mV (ketanserin) at 0.1 micromol/L, (-22.39+/-0.94) mV at 1 micromol/L, (-23.51+/-0.91) mV at 10 micromol/L, respectively (P<0.05, n=6). Characteristics of blockade were consistent with an open-state channel blockade, because the extent and rate of onset of blockade was voltage-dependent, increasing at more potentials even in the condition of leftward shift of activation curve. Meanwhile, in the different depolarization duration, the fractional blockade of end-pulse step current and peak tail current at 100 ms duration was significantly lower than that at 400 ms and 700 ms, which indicated that following the channel activation fractional blockade was enhanced by the activated channels. Ketanserin could also modulate the inactivation of HERG channel, which shifted the voltage-dependence of WT HERG channel inactivation curve from (-51.71+/-2.15) mV to (-80.76+/-14.98) mV (P<0.05, n=4). The S6 mutation, Y652A and Y652R, significantly attenuated the blockade by ketanserin. The IC50 value were (27.13+/-9.40) micromol/L and (20.20+/-2.80) micromol/L, respectively, increased by approximately 72-fold for Y652A and 53-fold for Y652R compared to that of WT HERG channel blockade [(0.38+/-0.04) micromol/L]. However, between the inhibitory effects of Y652A and Y652R, there was no significant difference. In conclusion, ketanserin blocks WT HERG currents in voltage- and concentration-dependent manner and preferentially blocks open-state HERG channels. Tyr-652 is one of the critical residues in the ketanserin-binding sites.

Effects of platelet activating factor on action potentials and potassium channels in guinea-pig ventricular myocytes / 生理学报

This study was designed to investigate the effects of platelet activating factor (PAF) on the action potential and potassium currents in guinea-pig ventricular myocytes. Whole cell patch clamp techniques were used. With 5 mmol/L ATP in the pipette electrode(mimic normal condition), 1 micromol/L PAF increased APD(90) from 225.8+/-23.3 to 352.8+/-29.8 ms (n=5, P<0.05), decreased I(K1) and I(K) tail currents from -6.1+/-1.3 to -5.6+/-1.1 nA (n=5, P<0.05) at -120 mV and from 173.5+/-16.7 to 152.1+/-11.5 pA (P<0.05, n=4) at +30 mV, respectively. But PAF had no effect on I(K1) at potentials within the normal range of membrane potentials (between -90 mV and +20 mV). In the contrary, without ATP in the pipette electrode by which I(K.ATP) was activated (mimic ischemic condition), 1 micro mol/L PAF shortened APD(90) from 153+/-24.6 to 88.2+/-19.4 ms (n=5, P<0.01). Incubation of myocytes with 1 micro mol/L glibenclamide, a blocker of I(K.ATP ) could restore prolongation of APD induced by PAF. In conclusion, in guinea-pig ventricular myocytes, with 5 mmol/L ATP in the pipette PAF could prolong APD partly due to the inhibition of I(K); while with 0 mmol/L ATP in the pipette, PAF could induce an activation of I(K.ATP), hence a decrease in APD. It is suggested that PAF may amplify the heterogeneity between ischemic and normal cardiac myocytes during ischemia /reperfusion, which may play a vital role in the pathogenesis of the arrhythmias induced by ischemia /reperfusion.

Chronic intermittent hypoxia decreases acute hypoxic inhibition of voltage-gated potassium channel in rat pulmonary arterial smooth muscle cells / 生理学报

For determination the ionic mechanisms of the hypoxic acclimatization at the level of channels, male Spradue-Dawley rats were divided into two groups: control normoxic group and chronic intermittent hypoxic group [O2 concentration: (10 +/-0.5)%, hypoxia 8 h a day]. Using whole cell patch-clamp technique, voltage-gated potassium channel currents (IK(V)) were recorded in freshly isolated pulmonary arterial smooth muscle cells (PASMCs) of rat with acute isolated method. The effect of acute hypoxia on IK(V) of PASMCs from chronic intermittent hypoxia group was investigated to offer some basic data for clarifying the ionic mechanisms of the hypoxic acclimatization. The results showed: (1) In control normoxic group, after acute hypoxia free-Ca(2+) solution, the resting membrane potential (Em) of PASMCs was depolarized significantly from -47.2+/-2.6 mV to -26.7+/-1.2 mV, and the IK(V) of PASMCs was decreased significantly from 153.4+/-9.5 pA/pF to 70.1+/-0.6 pA/pF, the peak current percent inhibition was up to (57.6+/-3.3)% at +60 mV, and current-voltage relationship curve shifted to the right. (2) In chronic intermittent hypoxic group, the IK(V) of PASMCs was decreased significantly by exposure to intermittent hypoxia in a time-dependent manner, appeared to start on day 10 and continued to day 30 (the longest time tested) of hypoxia, and current-voltage relationship curve shifted to the right in a time-dependent manner. (3) Compared with the control normoxic group, the percent IK(V) inhibition by acute hypoxia was significantly attenuated in the chronic intermittent hypoxia group and this inhibition effect declined with time exposure to hypoxia. The results suggest that K(V) inhibition was significantly attenuated by chronic intermittent hypoxia, and this effect may be a critical mechanism of the body hypoxic acclimatization.

Isolation and electrophysiological characteristics of embryonic cardiomyocytes in mice / 生理学报

To explore the electrophysiological characteristics of embryonic cardiomyocytes, single embryonic cardiomyocytes were obtained from mice at different periods by a collegenase B digestion approach, whole cell patch clamp recording technique was used to record I(f) and I(Ca-L), and spontaneous action potential was also recorded. The morphological and spontaneous contractile properties of the isolated cells appeared to be typical embryonic cardiomyocytes when the cells were assessed by phase-contrast microscope. Whole cell recording of isolated cells is easily performed by the whole cell patch clamp technique. Elelctrophysiological properties of I(f) and I(Ca-L) from embryonic cardiomyocytes have been proved to be similar to those from adult pacemaker cells or cardiomyocytes. The established method of isolation is simple, stable, effective and reliable. It allows to obtain as early as 8.5-day embryonic myocytes. The electrophysiological recording of embryonic cardiomyocytes will provide a useful model for exploring the electrophysiological characteristics of embryonic cardiomyocytes and the possible mechanism underlying some heart diseases.

Inhibitory effect of adrenomedullin on L-type calcium currents in guinea-pig ventricular myocytes / 生理学报

The effects of adrenomedullin (ADM) on the L-type calcium currents (I(Ca,L)) and the mechanism of the signal transduction process were studied. Enzymatically isolated guinea-pig ventricular myocytes were used to measure ICa,L with whole-cell patch-clamp techniques. ADM at the concentrations of 1-100 nmol/L decreased ICa,L in a dose-dependent manner (P<0.05). ADM22-52) (100 nmol/L), a specific ADM-receptor antagonist, completely abolished the ADM-induced inhibition of ICa,L. Pretreatment of the cells with H-89 (10 micromol/L), a specific PKA inhibitor, did not attenuate the effects of ADM. Intracellular application of 10 micromol/L PKC19-36), a specific PKC inhibitor, prevented the ADM-induced inhibition of the ICa,L, while the specific PKC activator PMA could mimic the effects of ADM on the ICa,L. PMA (1 micromol/L) decreased the ICa,L by 32.26+/-4.20%(P<0.05). These findings indicate that ADM can inhibit the ICa,L in guinea-pig ventricular myocytes, and the inhibition is mediated by the specific ADM-receptor and an activation of protein kinase C.